CHARACTERIZATION OF PURIFIED HUMAN HEMATOPOIETIC PROGENITOR CELLS OF BONE MARROW AND THEIR RECONSTITUTING ABILITY IN GENOGENEIC TRANSPLANTATION

我们在无血清培养中测试了早期造血因子对原始人骨髓祖细胞增殖的影响，并使用NOD/SCID/人类异种移植模型测试了纯化的原始人骨髓细胞的移植能力。基于CD34、Kit受体和CD38的表达，采用FAGS - Vantage对原始人骨髓细胞进行纯化。单细胞培养系统结果表明，钢铁因子（SF）、flk2/flt3配体（FL）和血小板生成素（TPO）在原始人造血祖细胞的存活和增殖中起重要作用。将原始骨髓细胞注入NOD/SCID小鼠体内，观察其移植能力。作为移植的标记，我们使用PCR分析了cd45阳性细胞和人类特异性DNA。第90天，在骨髓中可以看到新鲜细胞（CD34^+/Kit^+）的移植，而在SF、FL和TPO中培养的细胞则没有。为了评估培养细胞的移植能力，可能需要开发结合造血因子的培养系统。我们研究了4例SCN患者原始髓系祖细胞对造血因子的反应性。在血清补充和血清剥夺培养中，粒细胞集落刺激因子（G-CSF）的作用下，患者体内形成的粒细胞-巨噬细胞（GM）集落数量减少。结果表明，SCN患者的原始髓系祖细胞不仅对G-CSF，而且对早期或中间作用的造血因子SF、FL和IL-3的反应性存在缺陷。

We have tested the effects of early-acting hematopoietic factors on the proliferation of primitive human bone marrow progenitor cells in serum-deprived culture, and the engrafting capability of purified primitive human bone marrow cells using the NOD/SCID/human xenograft model. Primitive human bone marrow cells were purified by setting the FAGS Vantage based on the expression of CD34, Kit receptor and CD38. The results of single-cell culture system suggest that steel factor (SF), the ligand for flk2/flt3 (FL) and thrombopoietin (TPO) play important roles in survival and proliferation of primitive human hematopoietic progenitors.The engrafting capabilities of the primitive bone marrow cells were examined by injecting them into NOD/SCID mice. As marker for engraftment, we analyzed CD45-positive cells and human specific DNA using PCR The day-90 engraftment in the bone marrow was seen with fresh cells (CD34^+/Kit^+) but not with cells cultured in the presence of SF, FL and TPO.To evaluate the engrafting capability of cultured cells, the development of culture system using the combination of hematopoietic factors might be required.We studied the responsiveness of primitive myeloid progenitor cells to hematopoietic factors in four patients with SCN.The number of granulocyte-macrophage (GM) colonies formed in patients was decreased in response to granulocyte colony-stimulating factor (G-CSF) in both serum-supplemented and serum-deprived culture. The results suggest that primitive myeloid progenitor cells of patients with SCN have defective responsiveness to not only G-CSF but also the early- or intermediate-acting hematopoietic factors, SF, FL, and IL-3.